Cloning, Prokaryotic Expression And Polyclonal Antibody Preparation Of HCCR: A New Biomarker For Hepatocellular Carcinoma

Objective To clone the gene of a new biomarker for hepatocellualr carcinoma, construct the prokaryotic plasmid expressing truncated human cervical cancer oncogene (HCCR-1167-360) and develop the polyclonal antibody against HCCR.Methods HCCR-1167-360 was amplified by RT-PCR from HepG2 cells and cloned into the prokaryotic expression vector pRSET-B, then expressed in E.coli BL21(DE3) pLysS which was induced by IPTG. Recombinant protein was purified by Ni-NTA spin column and acryl amide gel electrophoresis, then its antigenicity was analyzed by Western blot. Polyclonal antibodies was developed by immunizing BALB/c mice with purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immuneosorbent assay, immuneohistochemical staining and Western blot analysis.Results1. The fragment of HCCR-1167-360 cDNA was obtained from HepG2 cells by RT-PCR.2. The prokaryotic expression plasmid pRSET-B.HCCR-1 was successfully constructed and confirmed by PCR, endonuclease digestion and direct sequencing.3. The recombinant protein HCCR-1 could be expressed in abundance in the form of inclusion bodies. We got the purified purpose protein of 27kD by Ni-NTA spin column kit. Western blot analysis showed that the His-tag was expressed correctly.4. According to the regular immunization method, high titer polyclonal antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein. Using ELISA and Western blot analysis, the titer of the polyclonal antibody was 1:16000 and 1:1000 respectively.Conclusions1. The truncated recombinant HCCR-1167-360 developed in this study had strong immunogenicity.2. Immunized with the purpose protein expressed, BALB/c mice generated polyclonal antibody against HCCR-1 successfully. The polyclonal antibody had both high sensitivity and high specificity.3. The anti-HCCR-1 polyclonal antibody developed can be applied in immuneohistochemical staining. Using the antibody positive signals were detected in human liver tissue.SignificanceImmunized with the truncated recombinant HCCR-1167-360 expressed, BALB/c mice generated polyclonal antibody against HCCR-1. The protein and the polyclonal antibody may provide an important tool for the research on biological function of HCCR, and for further evaluation its clinical significance on diagnosis of human hepatocellualr carcinoma.